Immunization with hepatitis C virus-like particles induces humoral and cellular immune responses in nonhuman primates

Abstract

We have previously reported the production of hepatitis C virus-like particles (HCV-LP) using a recombinant baculovirus containing the cDNA of the HCV structural proteins (core, E1, and E2). These particles resemble the putative HCV virions and are capable of inducing strong and broad humoral and cellular immune responses in mice. Here we present evidence on the immunogenicity of HCV-LP and the effects of novel adjuvant systems in a nonhuman primate model, the baboon. Three groups of four baboons were immunized with HCV-LP, HCV-LP and adjuvant AS01B (monophosphoryl lipid A and QS21), or HCV-LP and the combination of AS01B and CpG oligodeoxynucleotides 10105. After four immunizations over an 8-month period, all animals developed HCV-specific humoral and cellular immune responses including antibodies to HCV structural proteins and gamma interferon(+) (IFN-gamma(+))CD4(+) and IFN-gamma(+)CD8(+) T-cell responses. The immunogenicity of HCV-LP was only marginally enhanced by the use of adjuvants. The overall HCV-specific immune responses were broad and long lasting. Our results suggest that HCV-LP is a potent immunogen to induce HCV-specific humoral and cellular immune responses in primates and may be a promising approach to develop novel preventive and therapeutic modalities.

FIG. 1.

Anti-HCV antibody response in baboons immunized with HCV-LP. Shown are anti-E1 and -E2 (A) and anticore (B) antibody responses in individual animals immunized with HCV-LP alone (animals 1 to 4), HCV-LP + AS01 (animals 5 to 8), or HCV-LP + AS01/CpG (animals 9 to 12). These animals were immunized four times at weeks 0, 4, 8, and 34, and blood samples were collected prior to immunization and 4 weeks after each immunization. Anti-HCV antibodies at week 0, week 12, and week 38 are presented as signal to noise (S/N) ratio, and the cutoff value for positivity is 2. The S/N ratio was determined by using the mean value of serum samples diluted 1:100 from each animal. Kinetics of anti-E1 and -E2 (C) and anticore (D) antibody responses in animals immunized with HCV-LP or with vvHCV.S (positive control). The timing for each immunization is indicated by an arrow shown on the top of each graph. The mean anti-HCV values of serum samples diluted 1:100 from four baboons in each group are shown at weeks 0, 4, 8, 12, 38, and 68. The three groups of immunized animals are labeled HCV-LP, HCV-LP + AS01B, HCV-LP + AS01/CpG 10105, and the positive control is labeled vvHCV.S.

FIG. 2.

IFN-γ ELISPOT assay of T-cell responses in HCV-LP immunized animals. PBMC collected before immunization (week 0) and 4 weeks after the second to fourth immunizations (weeks 8, 12, and 38, respectively) were stimulated with recombinant HCV core protein, E1/E2 protein, HCV core overlapping peptides (OLPs), HCV E1 OLPs, and HCV E2 OLPs, respectively. Data are shown as HCV-specific IFN-γ SFU per 3 × 10⁵ cells for each individual baboon and represent the mean values of spots from duplicate tests after subtracting the background value (without antigen), which is typically less than 5 SFU.

FIG. 3.

CD8⁺ T-cell response obtained by intracellular cytokine staining (ICS) in HCV-LP-immunized animals. PBMC collected before immunization (week 0) and 4 weeks after the second to fourth immunizations (weeks 8, 12, and 38, respectively) were stimulated in vitro with HCV OLPs spanning core, E1, N-terminal E2(1), and C-terminal E2(2). Negative control without peptide and positive control with phorbol myristate acetate and ionomycin were included. Representative fluorescence-activated cell sorter histograms of baboons (percentage of IFN-γ⁺CD8⁺ cells shown in the right upper corner) from each immunized group after the fourth immunization are shown in panel A and the overall responses are summarized in panel B. For background determination, PBMC cultured without HCV-specific peptide stimulation were tested in parallel with those stimulated with HCV core and E peptides. The percentage of HCV core-specific or HCV E-specific IFN-γ⁺CD8⁺ cells from each group of animals was determined after subtracting the percentage of IFN-γ⁺CD8⁺ cells in the absence of HCV peptides from that in the presence of HCV peptides. The cutoff of the assay for each peptide pool is about 0.015%, which is set as twice the mean of multiple negative controls (without antigen).

FIG. 3.

CD8⁺ T-cell response obtained by intracellular cytokine staining (ICS) in HCV-LP-immunized animals. PBMC collected before immunization (week 0) and 4 weeks after the second to fourth immunizations (weeks 8, 12, and 38, respectively) were stimulated in vitro with HCV OLPs spanning core, E1, N-terminal E2(1), and C-terminal E2(2). Negative control without peptide and positive control with phorbol myristate acetate and ionomycin were included. Representative fluorescence-activated cell sorter histograms of baboons (percentage of IFN-γ⁺CD8⁺ cells shown in the right upper corner) from each immunized group after the fourth immunization are shown in panel A and the overall responses are summarized in panel B. For background determination, PBMC cultured without HCV-specific peptide stimulation were tested in parallel with those stimulated with HCV core and E peptides. The percentage of HCV core-specific or HCV E-specific IFN-γ⁺CD8⁺ cells from each group of animals was determined after subtracting the percentage of IFN-γ⁺CD8⁺ cells in the absence of HCV peptides from that in the presence of HCV peptides. The cutoff of the assay for each peptide pool is about 0.015%, which is set as twice the mean of multiple negative controls (without antigen).